Amygdalin potentiates the anti-cancer effect of Sorafenib on Ehrlich ascites carcinoma and ameliorates the associated liver damage

The burden of cancer diseases is increasing every year, therefore, the demands to figure out novel drugs that can retain antitumor properties have been raised. This study aimed to investigate the anti-tumor properties of amygdalin (Amy) against Ehrlich ascites carcinoma (EAC) bearing mice and its protective properties against liver damage. Amy and the standard anticancer drug Sorafenib (Sor) were given alone or in combination to Swiss albino female mice that had been injected with EAC cells. Biochemical parameters of liver function (AST, ALT, GGT, total protein, albumin), tumor volume, oxidative stress [malondialdehyde, (MDA)] and antioxidative [superoxide dismutase (SOD), and reduced glutathione (GSH)] markers were measured. The hepatic expression of the antioxidant-related gene [nuclear factor erythroid-2-related factor 2 (Nrf2)], the migration-related gene [matrix metalloprotease 9 (MMP9)], and the angiogenesis-related gene [vascular endothelial growth factor (VEGF)] were evaluated by qPCR. The results revealed that EAC-bearing mice treated with Amy and/or Sor showed a decrease in the tumor burden and hepatic damage as evidenced by (1) decreased tumor volume, number of viable tumor cells; (2) increased number of dead tumor cells; (3) restored the liver function parameters; (4) reduced hepatic MDA levels; (5) enhanced hepatic GSH and SOD levels; (6) upregulated expression of Nrf2; (7) downregulated expression of MMP9 and VEGF, and (8) improved hepatic structure. Among all treatments, mice co-treated with Amy (orally) and Sor (intraperitoneally) showed the best effect. With these results, we concluded that the Amy improved the antitumor effect of Sor and had a protective role on liver damage induced by EAC in mice.

. Mice initial body weights and change in body weight in different experimental groups. Data were presented as means ± SEM. Small (a-f) letters showed the marked change at P ≤ 0.05. The significant were expressed by dissimilar letters in the same column. Cnt Control, Amy Amygdalin, Sor Sorafenib, EAC Ehrlich ascetic carcinoma, IP Intraperitoneal, OS Per os (oral). www.nature.com/scientificreports/ Amy and/or Sor (IP and OS) restored the expression of these genes to levels comparable to the control groups with best improvement (highest Nrf2 and lowest MMP9 and VEGF) noticed in EAC + Amy + SorIP, followed by EAC + Amy + SorOS, then EAC + SorIP, EAC + SorOS, and finally EAC + Amy group (Fig. 2).
Histopathological examination. As shown in Fig. 3A, the liver of the control group showed normal hepatic architecture, central vein, portal vein area, polyhedral-shaped hepatocytes, and blood sinusoids. The liver of the Amy group showed congested central vein, mild dilation of blood sinusoids, mild degeneration of hepatocytes, and mild Kupffer cells activity (Fig. 3B). The liver of the SorIP group showed congested central Figure 1. Tumor ascitic fluid volume and count of live and/or dead EAC cells in EAC-bearing mice after treatment with amygdalin and/or sorafenib. Data were presented as means ± SEM (n = 7). Small (a-e) letters showed the marked change at P ≤ 0.05. The significant were expressed by dissimilar letters above columns with the same color.  (Fig. 3D). The liver of the Amy + SorIP group showed congested central vein, congestion, and dilation of blood sinusoids in addition to Kupffer cells activity (Fig. 3E). The liver of the Amy + SorOS group showed mild congestion of the central vein, mild dilation of blood sinusoids, and a moderate increase of Kupffer cells activity (Fig. 3F).
On the other hand, livers of untreated EAC-bearing mice (EAC group) showed aggregations of pleomorphic, hyperchromatic, and darkly basophilic cells (these cells were assumed to be EAC cells) in the perivascular area around the dilated and congested central vein as well as a notable area of hepatocellular necrosis (Fig. 3G). The five treated groups showed notable improvement in liver structure with less damage as compared to the EAC group. In the EAC + Amy group, the liver showed a moderate number of pleomorphic cells with moderate degeneration (Fig. 3H). The liver of the EAC + SorIP group showed a small focal area of pleomorphic cells with moderate degenerative changes (Fig. 3I). The liver of the EAC + SorOS group showed a moderate focal area of pleomorphic cells and mild dilation of some blood sinusoids (Fig. 3J). The liver of the EAC + SorOS group showed the smallest focal area of the pleomorphic cells with the mildest congestion in some blood sinusoids (Fig. 3K). The liver of the EAC + SorOS group showed small diffuse pleomorphic cells with mild vacuolar degeneration of hepatocytes (Fig. 3L).

Discussion
Mice injected with EAC cells and treated with Amy and/or Sor (IP or OS) showed a significant reduction in the ascitic fluid (tumor volume), the body weight, and viable tumor cell as compared to mice in the untreated EAC group. Among the five treated groups, the best antitumor effect (as revealed by less ascitic fluid, body weight, and viable EAC cells) was noticed in animals treated with both Amy and Sor with better effect for those injected IP by Sor. This antitumor effect could be attributed to enhanced phagocytes and cytokines production by Amy and/or Sor on EAC cells 18 . Our results also revealed that treatment with Amy and/or Sor not only significantly reduced the viable EAC cell count but also increased the count of non-viable cells. This infers that the antitumor action of Amy and Sor could be mediated through a direct inhibitory potential on the proliferation of tumor cells.
Hepatocytes are the main target for hepatic enzyme metabolism. Injury of these cells results in leakage of liver enzymes such as AST, ALT, and GGT from the liver into the circulation with an ultimate elevation of enzyme serum levels [19][20][21] . Hepatocytes are rigorously injured in animals with EAC cells 22 . This hepatic damage is accompanied by an increase in the levels of ALT and AST in the serum of EAC-bearing mice [22][23][24] . Consistent with these findings, we also found a significant elevation in ALT, AST, and GGT in the EAC group as compared to the control group. Reduced level of these hepatic damage enzymes in serum is associated with the antitumor potential of synthetic and natural anticancer compounds 19,25,26 . In agreement, we also found a significant reduction in the serum levels of these enzymes in EAC mice treated with Amy and/or Sor with best effect for animals treated with both Amy and SorIP. In line with our findings, another study reported that administration of Amy decreased the elevated ALT, AST, ALP, and GGT levels in N-nitrosodiethylamine-intoxicated rats 27 . Albumin and total proteins are other hepatic markers that are very important to follow up the progression of liver damage. We found a significant decrease in these two markers in EAC-bearing mice when compared to the control mice. Similarly, decreased levels in serum total protein and albumin were also observed in hepatic dysfunction cases 2,22 . Again, administration of Amy and/or Sor elevated these two markers in EAC-bearing mice with superior effect in co-treated mice, especially with Amy and SorIP. Similarly, Amy pretreatment increased albumin, total proteins in intoxicated rats 27 .
In the present study, treatment with Amy and/or Sor neutralized the effects induced by oxidative stress in EAC-bearing mice. This overall conclusion is based on our data which revealed that administration of Amy and/ or Sor inhibited the lipid peroxides (MDA) and increased the antioxidant markers (GSH and SOD). In support, Table 3. Hepatic oxidative (MDA) and antioxidative (GSH and SOD) parameters in EAC-bearing mice after treatment with amygdalin and/or sorafenib. Data were presented as means ± SEM. Small (a-g) letters showed the marked change at P ≤ 0.05. The significant were expressed by dissimilar letters in the same column.

Groups
MDA (nmol/g tissue) GSH (nmol/g tissue) SOD (U/g tissue) www.nature.com/scientificreports/ Amy increases the free radical scavenging activity through its stimulatory effect on GSH level and SOD activity in the liver of intoxicated rats 27 . The therapeutic potential of Amy is mainly attributed to its components that exhibit a wide range of biological effects including free radical scavenging 28 . Amy also improved the level of GSH in dimethylnitrosamine-induced liver fibrosis 27 . At a molecular level, mice treated with Amy and/or Sor showed a significant upregulation of the antioxidant-related Nrf2 gene, with best effect in mice co-treated with Amy and SorIP, as compared to the untreated EAC-bearing mice. Nrf2 is mandatory for defending the body against cancer 29 . www.nature.com/scientificreports/ It was found that tumor-induced angiogenesis is initiated by angiogenic cytokines such as basic fibroblast growth factor (bFGF) and VEGF that are expressed in the tumor itself 30 . The expression of VEGF is increased by the treatment of TGFβ, which is activated by MMP9 31 . Our data revealed a significantly upregulated expression www.nature.com/scientificreports/ of the migration-related MMP9 gene and the angiogenesis-related VEGF gene in the liver of the untreated EACbearing mice. Our results are consistent with El Bakary, et al. (32) who reported a significant elevation in the expressions of MMP9 and VEGF in EAC mice. This expression was downregulated following treatment with Amy and/or Sor, with best effect for Amy + SorIP. Histopathological examination showed aggregations of pleomorphic, hyperchromatic, and darkly basophilic cells assumed to be EAC cells in the perivascular area around the dilated and congested central vein and hepatocellular necrosis in the liver of the EAC group. Additionally, degenerative changes such as loss of histoarchitecture, due to microvascular fatty changes in addition to mild Kupffer cells activity were also observed. It was also reported that EAC cells can migrate from the peritoneal cavity and reach the liver causing liver injury 32 . EAC-bearing mice treated with Amy and/or Sor showed notable improvement in liver structure, especially in Amy + SorIP co-treated mice.

Conclusions
Treatment of EAC-bearing mice with Amy and/or Sor reduced tumor burden and lipid peroxidation, improved antioxidant status, and restored the damaged hepatocytes. Amy and SorIP are more effective in ameliorating the effect of liver damage in EAC-bearing mice. Amy could improve the antitumor effect of Sor on EAC. Hence, it could be utilized as an adjuvant for Sor during cancer therapy. Therefore, further studies are encouraged to create a novel strategy targeting cancer cells using Amy and Sor co-therapy.

Materials and methods
Animals. Research ethical approval was obtained from the research ethical committee, Faculty of Science, Tanta University, Egypt, as established by the institutional animal care and use committee (IACUC). All methods were completed in accordance with ARRIVE guidelines. The experiment was carried out on female Swiss albino mice weighing 20-25 g and of 10-12 weeks ages. Mice were maintained under standardized conditions. Mice were kept in a controlled temperature environment with a 24 h cycle. All mice were adapted to the place for two weeks before the start of the experiment. The animals were provided with a normal diet and water ad libitum. Four EAC-bearing mice at day 14 of EAC intraperitoneal (IP) injection were obtained from the Cancer Biology Unit, Cairo, Al-Kaser Al-Eini, Egypt. In our lab, the ascitic fluid containing EAC cells was maintained and propagated by serial aseptic IP transplantation in mice. Each mouse was injected with 200 mL of 1 × 10 6 EAC cells 33 . EAC cells filled the peritoneal cavity by fast division of cells, resulting in accumulation of ascitic fluid, and the animal could be died 17-18 days after EAC injection if did not receive appropriate treatment 3 .
Experimental design. Seventy-two mice were divided into 12 groups (n = 6/group). Normal (control) group: mice were injected IP with normal saline (0.9% w/v, 300 µl/mouse). Amy group: mice were administrated amygdalin. SorIP group: mice were IP treated with sorafenib. SorOS group: mice were treated orally with sorafenib. Amy + SorIP group: mice were administered amygdalin and injected IP with sorafenib. Amy + SorOS group: mice were orally given amygdalin and sorafenib. EAC group: mice were injected IP by 200 mL of 1 × 10 6 EAC cells and left for 14 days without treatment. EAC + Amy group: animals were injected once with EAC cells and 24 h later they were treated with amygdalin. EAC + SorIP: mice were inoculated with EAC cells and 24 h later they were IP injected with sorafenib. EAC + SorOS: mice were inoculated once with EAC cells and 24 h later they were orally administered sorafenib. EAC + Amy + SorIP: mice were inoculated once with EAC and 24 h later they were co-treated with amygdalin and sorafenib (IP). EAC + Amy + SorOS group: mice were inoculated with EAC and 24 h later they were co-treated with amygdalin and sorafenib. All treatments were given daily for 14 days and the doses of amygdalin (300 mg/kg mouse) and sorafenib (30 mg/kg mouse for both IP and OS) were chosen based on a pilot study with aid of previous studies 34,35 . Amygdalin (Vitamin B17) and sorafenib were purchased from Sigma-Aldrich and BAYER companies, respectively. The animals were weighed at the beginning of the experiment (initial body weight, g), at the end of the experiment (final weight, g) and the mean body weight change (g) was then calculated by subtracting the initial weight from the final weight. It is well-known that changes in body weight of EAC-bearing mice is an additional indirect measure of changes in tumor mass in these animals.

Sampling.
At the end of the experimental period (2 weeks), overnight fasted rats have sacrificed for 24 h.
After the last treatment, blood samples were centrifuged in clean glass tubes for 15 min at 3000×g to get clear, non-hemolyzed sera. Eppendorf tubes with labels were immediately shipped to − 20 °C; the sera were frozen for biochemical analysis. After euthanization by exsanguination, livers were immediately removed and some specimens were fixed in 10% formalin (pathological investigation), and the others were either homogenized (biochemical assay) or frozen in − 70 °C (RNA extraction).
Tumor (ascitic fluid) volume and EAC count. At the end of the experiment (day 14), ascitic fluid containing EAC cells was withdrawn from the peritoneal cavity of each mouse before they were dissected. A graduated centrifuge tube was used to measure how much ascitic fluid was collected. Following the suspension of EAC cells in sterile isotonic saline, a Neubauer hemocytometer was used to count the total, viable and non-viable EAC cells 33 . Assessment of biochemical parameters. The serum level of liver damage enzymes [aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT)], albumin, and total proteins were measured using commercially available kits (Biomed Diagnostics, Cairo, Egypt). Liver homogenates were pre- www.nature.com/scientificreports/ pared as previously described 36 . The hepatic levels of lipid peroxidation marker malondialdehyde (MDA) and the antioxidant markers reduced glutathione (GSH) and superoxide dismutase (SOD) were measured colorimetrically using kits purchased from Biodiagnostics and as previously described 37,38 . Real-time PCR. Real-time PCR was used to determine changes in the relative expression of Nrf2, MMP9, and VEGF genes in liver specimens of all groups. Total RNA was first isolated and then reverse transcribed into cDNA by kits purchased from Thermo Scientific, USA (# K0731 and #EP0451, respectively). The sequences of primers were as following: F: 5′ CAC ATC CAG ACA GAC ACC AGT 3′ and R: 5′ CTA CAA ATG GGA ATG TCT CTG C 3′ for Nrf2; F: 5′ TCG AAG GCG ACC TCA AGT G 3′ and R: 5′ TTC GGT GTA GCT TTG GAT CCA 3′ for MMP9; F: 5′ GAT CAT GCG GAT CAA ACC TCACC 3′ and R: 5′ CCT CCG GAC CCA AAG TGC TC 3′ for VEGF; F: 5′ CAT GGA TGA CGA TAT CGC T 3′ and R: 5′ CAT GAG GTA GTC TGT CAG GT 3′ for β actin (internal control). Thermal and melting curve conditions were done as previously detailed [39][40][41] . The fold change in gene expression was determined using the 2 −∆∆Ct method.
Histopathological investigation. Liver tissues were dehydrated in ascending series of ethanol, cleared in xylene, embedded in paraffin wax, sectioned at 5 µm thickness by a microtome, stained with eosin and hematoxylin, examined, and photographed under a light microscope to detect histopathological changes.
Statistical analysis. GraphPad Prism 5.0 was used to analyze the data. The experimental results were expressed as mean ± standard error mean (SEM). Data were assessed by one-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons test. Values for which P < 0.05 were considered statistically significant.
Institutional review board statement. The study was conducted according to the guidelines of ARRIVE and approved by Ethical committee at the Faculty of Science, Tanta University.

Data availability
The data presented in this study are available on request from the corresponding author.